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Biomedical subjects

S Inouye

Publications and source records attributed to S Inouye.

At least 415 records · Page 23Linked to original sources
PubMed1985

Comparative bactericidal and morphological effects of five cephamycins on cells of three gram-negative bacilli at decreasing drug concentrations.

The bactericidal and morphological effects under drug-decreasing conditions of cefminox (MT-141), cefoxitin, cefmetazole, cefotetan and latamoxef were compared on Klebsiella pneumoniae PCl-602, Escherichia coli No. 29 and Serratia marcescens No. 1 cells. A new drug-decreasing method was developed by the use of an antibiotic removal device. Cefminox displayed an earlier onset and a higher rate of bactericidal action than the other cephamycins against the test organisms, although the minimum inhibitory concentrations of cefminox were similar to or higher than those of the other drugs. Morphologically, cefminox caused rapid lysis of the cells without filamentation, whereas the reference cephamycins caused mainly elongation of the cells under the test conditions. Frequent formation of multiple bulges on cells exposed to cefminox was observed in an isotonic medium.

Cephamycins↗
PubMed1984

Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid.

The breakage of double-strand (ds) DNA by 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid (LAHPO) was investigated by agarose gel electrophoresis of supercoiled pBR322 DNA and the site of cleavage on the DNA molecule was determined by the method of DNA sequence analysis using 3'-end and 5'-end-labeled DNA fragments as substrates. LAHPO caused cleavage at the position of guanine nucleotide in dsDNA. LAHPO caused dsDNA breaks at specific sites, but linoleic acid (LA) and 13-L-hydroxy-cis-9,trans-11-octadecadienoic acid (LAHO) have no such effects on dsDNA. The active oxygen atom of the hydroperoxy group of LAHPO was perhaps responsible for the site-specific cleavage of dsDNA.

Animals↗
PubMed1984

Verification of protein sequence by fast atom bombardment mass spectrometry. Amino acid sequence of protein S, a development-specific protein of Myxococcus xanthus.

A mass spectrometric method was applied to protein S, a development-specific protein of Myxococcus xanthus, in order to verify the amino acid sequence deduced from the nucleotide sequence of its gene. On examining proteolytic digests of the protein by fast atom bombardment mass spectrometry without separation of individual peptides, signals corresponding to individual peptides were observed in the mass spectra. The mass values of the observed signals were correlated to the theoretical mass values calculated from the predicted amino acid sequence, thereby identifying the peptides and proving the accuracy of the sequence. The previous finding that protein S is the product of the second gene of the two tandemly repeated genes on the M. xanthus chromosome ( Inouye , S., Franceschini , T., and Inouye , M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833) is now unambiguously confirmed.

Amino Acid Sequence↗
PubMed1984

Effects of replacing serine and threonine residues within the signal peptide on the secretion of the major outer membrane lipoprotein of Escherichia coli.

We have investigated the importance of serine and threonine residues within the signal peptide in the secretion and processing of the major outer membrane lipoprotein precursor prolipoprotein in Escherichia coli. This was accomplished by systematically replacing these residues with alanine utilizing oligodeoxyribonucleotide-directed mutagenesis. The results demonstrated that the replacement of serine 15 but not threonine 16 alone caused an initial accumulation of membrane-bound unmodified prolipoprotein. In addition, replacement of both serine 15 and threonine 16 resulted in a greater accumulation of this membrane-bound precursor. The accumulated prolipoprotein could be matured to lipoprotein in a quantitative manner, and this process was inhibited by globomycin and carbonyl cyanide m-chlorophenylhydrazone. These results will be discussed in terms of the contribution that serine and threonine have in determining the overall secondary structure of the signal peptide and its importance in secretion and/or processing.

Bacterial Outer Membrane Proteins↗
PubMed1984

Identification of a development-specific promoter of Myxococcus xanthus.

In the chromosome of Myxococcus xanthus, two homologous genes for protein S, a development-specific protein, are tandemly repeated with a 1.4 X 10(3) base-pair sequence between the two genes. Two synthetic oligodeoxyribonucleotides were used as specific probes for individual transcripts from the upstream gene 1 and the downstream gene 2, respectively. The gene 2 transcript was detected only during developmental growth, while the gene 1 transcript was not detected during developmental or vegetative growth. The major initiation site for the gene 2 transcription was determined to be 51 bases upstream of the initiation codon for gene 2. The development-specific promoter of gene 2 was identified; it shows some homologies to the Escherichia coli promoter structures.

Bacterial Proteins↗
PubMed1984

Effects of mutations at glycine residues in the hydrophobic region of the Escherichia coli prolipoprotein signal peptide on the secretion across the membrane.

Each of the 2 glycine residues in the hydrophobic region of the prolipoprotein signal peptide of Escherichia coli was systematically deleted or substituted with a valine residue by oligonucleotide-directed site-specific mutagenesis. Functional analysis of four such mutants as well as four double mutants, resulting from combinations of any two of the single mutations, revealed that (a) glycine residues at positions 9 and 14 could be replaced individually or at the same time with a valine residue without affecting the secretion of prolipoprotein; (b) the deletion of glycine at position 9 had no effect on the secretion of prolipoprotein whereas, when glycine at position 14 was deleted, the glyceride modification and the processing of the mutant prolipoprotein occurred at a much slower rate at 42 degrees C than those of the wild type prolipoprotein; and (c) the effects of deleting glycine at position 14 could be suppressed by the deletion of glycine at position 9, which resulted in shortening the hydrophobic region of the prolipoprotein signal peptide by 2 amino acid residues. These results indicate that the hydrophobic region of the prolipoprotein signal peptide has remarkable flexibility in terms of the relationship between its primary structure and function in protein secretion.

Amino Acid Sequence↗
PubMed1984

Multicopy single-stranded DNA isolated from a gram-negative bacterium, Myxococcus xanthus.

A gram-negative bacterium, Myxococcus xanthus, was found to contain 500 to 700 copies per chromosome of a short single-stranded linear DNA fragment. When this DNA (multicopy single-stranded DNA; msDNA) labeled at the 5' end with kinase was used as a probe against total chromosomal blots, it hybridized to unique high molecular weight bands, which were cloned and sequenced. Labeling of msDNA was also possible using the Klenow fragment of DNA polymerase I as well as terminal deoxynucleotidyl transferase, permitting direct sequencing. The 5' end of msDNA was found to be primed by a short RNA segment. The DNA portion of msDNA consisted of 163 bases. Exact correspondence was seen between the msDNA sequence and the sequence of a chromosomal clone. An elaborate secondary structure is postulated for the msDNA sequence. A similar satellite DNA was also found in another myxobacterium, Stigmatella aurantiaca.

Base Sequence↗
PubMed1984

Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida.

The transcription initiation site of the xylDEGF operon on the TOL plasmid of Pseudomonas putida mt-2 was determined in P. putida and in Escherichia coli by S1 nuclease and reverse transcriptase mapping. The induced synthesis of mRNA started at the same start point in both P. putida and E. coli, although the amount of mRNA in E. coli cells was less than that in P. putida. The nucleotide sequence of the region surrounding the start point was also determined. The ribosome-binding site (RBS) complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli preceded the predicted start codon for the xylD gene. The consensus nucleotide sequence for E. coli promoters was not found in the region preceding the transcription start point. On the other hand, the sequences of the "-10" and the "-35" regions of the xylDEGF operon revealed some homology with the respective, previously determined sequences of the xylABC operon of the TOL plasmid.

Base Sequence↗
PubMed1984

Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida.

The xylABC operon on the TOL plasmid directs the synthesis of enzymes for conversion of toluene to benzoate and is positively controlled by the regulatory gene xylR. In the study here the nucleotide sequence was determined for the regulatory region of this operon. The in vivo transcription initiation site of the operon was determined by S1 nuclease and reverse transcriptase mapping. RNA was prepared from m-methylbenzyl alcohol-induced cells of Pseudomonas putida and Escherichia coli carrying pTN2, a derivative of the TOL plasmid containing the structural and regulatory genes of the entire toluene-degrading pathway. The amount of E. coli mRNA was estimated to be only 10% of that of P. putida mRNA. Consensus sequences of the -10 region (Pribnow box) and the -35 region (RNA polymerase recognition site) in E. coli genes were not found in the region preceding the transcription initiation site, whereas a sequence complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli existed in front of the predicted start codon of the xylA gene. These results explain the inefficient expression of TOL genes in E. coli.

Base Sequence↗
PubMed1984

"Original antigenic sin" phenomenon in experimental flavivirus infections of guinea pigs: studies by enzyme-linked immunosorbent assay.

Guinea pigs were sequentially infected with two closely related flavivituses (Japanese encephalitis and West Nile viruses), and their antibody responses were studied both by enzyme-linked immunosorbent assay (ELISA) and hemagglutination-inhibition (HI). The results by ELISA always showed "original antigenic sin" responses: antibody activity of sera after the second infection was higher to the first infecting virus than to the second infecting virus. However, the results obtained by HI were variable.

Animals↗
PubMed1984

In vitro and in vivo antibacterial activities of MT-141, a new semisynthetic cephamycin, compared with those of five cephalosporins.

The in vitro and in vivo antibacterial activities of MT-141 were compared with those of cefoxitin, cefmetazole, moxalactam, cefotaxime, and cefoperazone. The MICs of MT-141 for 90% of bacterial isolates were lower than the reference drugs against clinical isolates of Campylobacter jejuni, Clostridium difficile, and Bacteroides fragilis, whereas against clinical isolates of other gram-positive, gram-negative, and anaerobic bacteria, the MICs of MT-141 were similar to or higher than those of the reference drugs. In contrast, the bactericidal activity of MT-141 after 6- and 24-h exposures was superior to all of the reference drugs against 9 to 10 of the 12 bacterial strains studied, including Escherichia coli, Klebsiella pneumoniae, Salmonella enteritidis, indol-positive Proteus species, Serratia marcescens, Yersinia enterocolitica, Pseudomonas cepacia, and Clostridium perfringens. In the treatment of systemic infections in mice, MT-141 was again superior against 9 of the 12 strains tested, showing a good correlation with the bactericidal activity. It was found that the 50% effective doses of the six cephalosporins studied correlated better with the MBCs than with the MICs. As the serum levels of MT-141 in mice after subcutaneous administration were similar to those of the reference drugs, it was concluded that the bactericidal activity of MT-141 was a dominant factor in its in vivo activity.

Animals↗
PubMed1984

Effects of deletion of the gene for the development-specific protein S on differentiation in Myxococcus xanthus.

A deletion mutation of the gene for protein S (tps), a development-specific protein of Myxococcus xanthus, was constructed. No significant differences in the process of fruiting body formation or the yield of myxospores were observed between mutant and wild-type cells. On the other hand, when the tps gene was deleted together with a 2.0-kilobase sequence including the ops gene immediately upstream of the tps gene, fruiting body formation was substantially delayed, and the yield of myxospores was reduced. These results indicate that protein S is not essential for differentiation of M. xanthus, whereas a gene product(s) coded from the sequence upstream of the tps gene appears to be required for normal fruiting body formation.

Bacterial Proteins↗
PubMed1984

Penicillin-insensitive incorporation of D-amino acids into cell wall peptidoglycan influences the amount of bound lipoprotein in Escherichia coli.

Certain D-amino acids, such as D-methionine and D-cystine, were incorporated into cells of Escherichia coli under conditions inhibiting protein and cell wall synthesis. Part of the radioactivity of D-14C-amino acids incorporated into the cells was found in the isolated cell wall peptidoglycan. A covalent linkage between the amino group of the D-amino acids and the peptidoglycan was presumed to be the main cause of the binding of the D-amino acids to peptidoglycan, because the amino group of the D-amino acids in the incorporation product was substituted. Whether the carboxyl terminus was substituted was unknown. The formation of the D-amino acid-peptidoglycan linkage was insensitive to beta-lactam antibiotics such as benzylpenicillin and ampicillin (500 micrograms/ml) and therefore was not due to the reaction of DD-transpeptidation which is involved in the biosynthesis of peptidoglycan. The D-amino acids also strongly inhibited the formation of peptidoglycan-bound lipoprotein in the E. coli cells. The results may suggest the correlation between binding of D-amino acid to peptidoglycan and inhibition of formation of the bound form of lipoprotein.

Amino Acids↗
PubMed1984

Efficient coating of the solid phase with rotavirus antigens for enzyme-linked immunosorbent assay of immunoglobulin A antibody in feces.

Sensitivity of the enzyme-linked immunosorbent assay for detecting serum antibodies to rotavirus was greatly enhanced when rotavirus particles were fragmented by chaotropic agents (NaSCN or guanidine hydrochloride) before adsorption of the antigens to the solid phase. For detecting fecal antibodies, the addition of fetal calf serum to the diluent was further needed to protect the antigens from the proteolytic activity of feces. With this technique, we readily detected immunoglobulin A antibody in feces from infantile gastroenteritis patients. Rate-zonal centrifugation of feces revealed that immunoglobulin A antibody activity sedimented with two peaks: one at 11S with a secretory component and another sedimenting slower than 7S, presumably as Fab portions.

Antibodies, Viral↗
PubMed1984

Changes in antibody avidity after virus infections: detection by an immunosorbent assay in which a mild protein-denaturing agent is employed.

In titrating serum immunoglobulin G antibody to viruses by enzyme-linked immunosorbent assay, we used two rows of wells for serial twofold dilutions of the serum; in one row, a low concentration of a protein denaturant, 0.5 or 1.0 M guanidine hydrochloride, was added to the diluent so that the binding of low-avidity antibodies to viral antigens on the solid phase was inhibited. We then compared the antibody titration curves obtained in the two rows. We found that the addition of the reagent resulted in a parallel leftward shift of the curves and that the extent of the shift was greater in early than in late sera from all of the three infections studied (Japanese encephalitis virus, rotavirus, and rubella virus infections). This procedure may be useful for estimation of the avidity of antibody in serum and, with further evaluation, may prove to be applicable to single-serum diagnosis of virus infections.

Adolescent↗
PubMed1984

Studies on a new nucleoside antibiotic, dapiramicin. II. Isolation, physico-chemical and biological characterization.

New nucleoside antibiotics, dapiramicins A and B produced by a Micromonospora sp. SF-1917, have been isolated by column chromatography on Diaion HP-20 and silica gel. Physico-chemical properties suggested that they are disaccharide nucleosides. Dapiramicin A underwent epimerization, under acidic condition, into epidapiramicin A. Although dapiramicin A generally exhibits no in vitro activity, it is very effective against the sheath blight of rice plants caused by Rhizoctonia solani in a green house test.

Aminoglycosides↗
PubMed1984

Structure-activity relationships on the terminal D-amino acid moiety of a novel cephamycin MT-141.

The effect of chemical modification of the D-amino acid function, which represents the C-7 beta substituent of cephamycin MT-141 on in vitro antibacterial activity was examined. MT-141 was more active on Gram-negative organisms than Gram-positive ones. It showed strong bacteriolytic activity on Gram-negative organisms. Lysis of Escherichia coli K-12 strain JE1011 treated with a low concentration of this antibiotic was preceded by frequent formation of multiple bulges from the cells. Amidation or decarboxylation, removing the acidic function from the D-amino acid of MT-141, resulted in an increase in activity against Gram-positive bacteria, and a decrease against Gram-negative ones. Cells treated with the amide or the decarboxylate did not form multiple bulges but formed single bulges. N-Acetylation of the D-amino acid moiety removing the basic function, caused a marked drop in activity against both Gram-positive and Gram-negative bacteria. The bacteriolytic activity on E. coli was reduced, and cells treated with the N-acetate became filamentous. Conversion of the D-amino acid function of MT-141 to the L configuration caused a moderate drop in activity against both Gram-positive and Gram-negative organisms. Both bacteriolytic and bactericidal activities against E. coli were reduced in the L-congener. Cefoxitin, cefmetazole and latamoxef used as reference antibiotics were less active than MT-141 in the bactericidal activity against E. coli, and induced single bulge formation or filamentation of the cells around MIC levels. Cell-surface permeability, stability to beta-lactamases, and binding affinity to PBPs of E. coli did not differ between MT-141 and its derivatives.

Bacteria↗